Abstract
Holo- and apocytochrome P450cam were studied by differential scanning calorimetry (DSC), limited proteolysis, second-derivative spectroscopy, circular dichroism, and size-exclusion chromatography. The holoprotein shows three folding units (domains) in DSC. The prosthetic group is related to the most unstable domain, which has a thermal transition at 41.9 degrees C. Compared with the holoprotein, apocytochrome P450cam has a reduced helix content. The protein is compact as judged by the Stokes radius and is still able to undergo a two-state transition. However, the enthalpy change at thermal melting is reduced from 980 kJ/mol for the holoprotein to 135 kJ/mol for the apo form. Parts of the molecule have a destabilized tertiary structure. This is indicated by second-derivative spectroscopy, circular dichroism in the near-ultraviolet region, and a high susceptibility to proteolytic digestion. Apocytochrome P450cam is considered a native protein with the extremely low stability of delta G = 7.5 kJ/mol, thus showing at the same time intermediate-like properties. The importance of the properties for in vivo folding are discussed.
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